Our goal is to understand in molecular detail the mechanisms of transcription, replication and packaging of the three double- stranded RNA chromosomes of the bacterial virus 06, using our characterized cDNA clones of the large segment that encodes these functions. Specific objectives for the continuation period are: 1. Complete assessment of functional activity of expressed products of 4 06 L segment genes. We have shown that P7, P2, and P4 are active. Pl appears to be detrimental when expressed in current constructs. 2. Complete characterization of a T7 polymerase/T7 promoter based regulated broad host range vector expression system, developed in conjunction with the complementation studies, by analysis of lacZ expression in several different hosts. 3. Overexpression of the four 06 L segment genes. Current constructs fail to overproduce these proteins so we will systematically try to discover whether the cause is at the transcriptional or translational level and remedy the situation to provide a convenient source for purification of the proteins. 4. One or more of the functional, overexpressed 06 L segment proteins will be purified by standard methods using virion or nucleocapsid antibodies, specific RNA binding or radioactive tracer proteins to monitor progress. 5. Continue experiments to analyze 06 replication in vivo and in vitro to determine the RNA template, the 06 proteins involved, and the specific requirements for initiation and elongation. Preliminary experiments suggest that we have separated replication from transcription in infected cell extracts. 6. A similar analysis of ts mutants defective in replication or transcription may yield a complementation assay for protein purification.